INTERNATIONAL JOURNAL OF ONCOLOGY 54: 1409-1421, 2019 Downregulation of protein disulfide‑isomerase A3 expression inhibits cell proliferation and induces apoptosis through STAT3 signaling in hepatocellular carcinoma RYOTA KONDO1,2, KOUSUKE ISHINO1, RYUICHI WADA1, HIDEYUKI TAKATA2, WEI-XIA PENG1, MITSUHIRO KUDO1, SHOKO KURE1, YOHEI KANEYA1,2, NOBUHIKO TANIAI2, HIROSHI YOSHIDA2 and ZENYA NAITO1 Departments of 1Integrated Diagnostic Pathology and 2Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo 113-8602, Japan Received September 26, 2018; Accepted December 14, 2018 DOI: 10.3892/ijo.2019.4710 Abstract. Protein disulfide-isomerase A3 (PDIA3) is a proteins (Bcl-2-like protein 1, induced myeloid leukemia cell chaperone protein that modulates folding of newly synthesized differentiation protein Mcl-1, survivin and X-linked inhibitor glycoproteins and responds to endoplasmic reticulum (ER) of apoptosis protein). In addition, PDIA3 knockdown provided stress. Previous studies reported that increased expression little inhibitory effect on cell proliferation in HCC cell lines of PDIA3 in hepatocellular carcinoma (HCC) is a marker treated with AG490, a tyrosine-protein kinase JAK/STAT3 for poor prognosis. However, the mechanism remains poorly signaling inhibitor. Finally, an association was demonstrated understood. The aim of the present study, therefore, was to between PDIA3 and P-STAT3 expression following understand the role of PDIA3 in HCC development. First, immunostaining of 35 HCC samples. Together, the present immunohistochemical staining of tissues from 53 HCC data suggest that PDIA3 promotes HCC progression through cases revealed that HCC tissues with high PDIA3 expression the STAT3 signaling pathway. exhibited a higher proliferation index and contained fewer apoptotic cells than those with low expression. In addition, the Introduction knockdown of PDIA3 significantly inhibited cell proliferation and induced apoptosis in HCC cell lines. These results suggest Hepatocellular carcinoma (HCC) is one of the most common that PDIA3 regulates cell proliferation and apoptosis in HCC. causes of cancer-associated mortalities worldwide (1). The An examination of whether PDIA3 knockdown induced principal treatment for HCC is surgical resection or liver apoptosis through ER stress revealed that PDIA3 knockdown transplantation (2). However, in the period between 2001 did not increase ER stress marker, 78 kDa glucose-regulated and 2008, the cumulative recurrence rate following complete protein, in HCC cell lines. Furthermore, the association resection of the tumor remained high (40-60% after 3 years) (3-5). between PDIA3 and the signal transducer and activator of Surgical treatment is often unavailable for patients with transcription 3 (STAT3) signaling pathway were investigated HCC who were diagnosed at an advanced stage. Multikinase in vitro and in vivo. Immunofluorescence staining and inhibitors (sorafenib, regorafenib and lenvatinib) are approved co-immunoprecipitation experiments revealed colocalization for the systemic treatment of inoperable HCC. However, such and binding, respectively, of PDIA3 and STAT3 in HCC drugs prolong the survival of patients with inoperable HCC for cell lines. The knockdown of PDIA3 decreased the levels of only a few months (6,7). Therefore, it is necessary to investigate phosphorylated STAT3 (P-STAT3; Tyr705) and downstream novel therapeutic targets for this disease. proteins of the STAT3 signaling pathway: The anti-apoptotic The signal transducer and activator of transcription 3 (STAT3) pathway is one of the major signaling pathways that promote tumor progression in HCC (8). This pathway is stimulated by inflammatory cytokines and growth factors (9-11). Activation of STAT3 through phosphorylation Correspondence to: Professor Zenya Naito, Department of Integrated Diagnostic Pathology, Nippon Medical School, of tyrosine 705 leads to the formation of dimers that 1-1-5 Sendagi, Tokyo 113-8602, Japan subsequently move from the cytosol to the nucleus to bind E-mail: [email protected] DNA, and thereby regulate gene expression and promote cell proliferation and survival (9-11). In the case of the Key words: apoptosis, hepatocellular carcinoma, proliferation, liver, the STAT3 signaling pathway is activated by chronic protein disulfide-isomerase A3, signal transducer and activator of hepatitis (hepatitis B or C infections, alcoholic hepatitis and transcription 3 non-alcoholic steatohepatitis) (11). It has been demonstrated that activated STAT3 is associated with tumor invasiveness, metastasis and poor prognosis in HCC (12-14). 1410 KONDO et al: PDIA3 AND STAT3 IN HEPATOCELLULAR CARCINOMA Protein disulfide‑isomerase A3 (PDIA3), also known as The THLE-2 cell line was maintained in Bronchial Epithelial endoplasmic reticulum (ER) resident protein 57 or 58 kDa Cell Growth Medium (BEGM; Lonza Group Ltd., Basel, glucose-regulated protein, is a thiol oxidoreductase with Switzerland) without gentamycin/amphotericin and epinephrine protein disulfide isomerase activity. PDIA3 modulates the but with added 5 ng/ml epidermal growth factor (Corning, folding of newly synthesized glycoproteins and misfolded Inc., Corning, NY, USA), 70 ng/ml phosphoethanolamine proteins in the ER (15). This protein also protects cells (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and from ER stress-induced apoptosis (15). Furthermore, it has a 10% FBS. The THLE-2 cells were cultured in RPMI-1640 variety of functions in the cytosol and nucleus (15). In several medium with 10% FBS for 7 days at 37˚C in a humidified types of cancer, PDIA3 forms a complex with STAT3 in the 5% CO2 atmosphere prior to protein extraction. A JAK/STAT3 nucleus (16-18). A high frequency of PDIA3-STAT3 complex signaling inhibitor, AG490 (Merck KGaA), dissolved in formation is a marker of poor prognosis and increases resistance dimethylsulfoxide (DMSO; Wako Pure Chemical Industries, to radiotherapy in laryngeal cancer through modulation of Ltd., Osaka, Japan), was used for the suppression of STAT3 STAT3 activity (19). We have previously reported that the signaling at 25 or 100 µM in cell growth or western blotting, PDIA3 expression levels in HCC tissues are higher than those respectively. in adjacent non-cancerous tissues, and that they are associated with overall survival time (20). However, the biological role of PDIA3 knockdown. Short-interfering RNAs (siRNAs) were PDIA3 in HCC remains unclear. purchased from Thermo Fisher Scientific, Inc. The two PDIA3 The aim of the present study was to understand the role of siRNAs used were designated PDIA3 si-1 and PDIA3 si-2 and PDIA3 in HCC. PDIA3 expression levels, the effects of PDIA3 known as Silencer® Select Pre-designed siRNA cat. no. 4392420, knockdown and the association between PDIA3 and STAT3 in ID s6227 (5'-GGAAUAGUCCCAUUAGCAAtt-3') and ID HCC were examined. The expression of PDIA3 in HCC tissues s6229 (5'-GCAACUUGAGGGAUAACUAtt-3'), respectively. was associated with cell proliferation, survival and expression of Silencer® negative control #1 siRNA (cat. no. 4390844) was phosphorylated STAT3 (P-STAT3) in HCC. PDIA3 knockdown used as a negative control (Ctrl si). The transfection of the in HCC cell lines inhibited cell proliferation and induced siRNA was performed using Lipofectamine® RNAiMAX apoptosis by suppression of the STAT3 signaling pathway. Reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according In the presence of the tyrosine-protein kinase JAK/STAT3 to the manufacturer's protocol. The optimal concentration of signaling inhibitor AG490, PDIA3 knockdown provided little siRNA for use in transfections was 5 nM. The medium was additional inhibition of cell growth. These data suggest that refreshed 24 h after cell seeding and the cells were cultured PDIA3 promotes tumor development in patients with HCC for a further 48 and 72 h for the apoptosis and cell cycle through the STAT3 signaling pathway. assay and western blotting, respectively. The cell transfection efficiency was measured by reverse transcription‑quantitative Materials and methods polymerase chain reaction (RT-qPCR) and western blotting, as described below. Clinical samples. A total of 53 patients with HCC who underwent hepatectomy without preoperative therapy at the RT‑qPCR assay. A total of 2.5x105 cells were seeded in 60-mm Nippon Medical School Hospital (Tokyo, Japan) between dishes and cultured for 48 h. Total RNA was extracted using January 2016 and February 2018, were enrolled in this the NucleoSpin RNA kit (Takara Bio Inc., Otsu, Japan), and study. The tumor and adjacent normal tissues from these 1 µg of total RNA was used for reverse transcription using patients were fixed with 10% formalin at room temperature the SuperScript VILO cDNA Synthesis kit (Thermo Fisher for 24 h within 2 h of resection and embedded in paraffin. Scientific, Inc.) following the manufacturer's protocol. The Among them, 35 HCC samples were formalin‑fixed within reaction conditions were as follows: 25˚C for 10 min, 42˚C for 30 min of resection and were used for immunostaining of 60 min and 85˚C for 5 min. The qPCR was performed for PDIA3 P-STAT3. The baseline characteristics of the patients are and 18S rRNA (as an internal standard) using the StepOnePlus summarized in Table I. This study was conducted according Real-Time PCR system with TaqMan probes and primers (18S, to the Declaration of Helsinki and the Japanese Society of cat. no. Hs 03928990_g1; PDIA3, cat. no. Hs 04194196_g1) (all Pathology, and was given official approval by the